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Structure of and kinetic channelling in bifunctional dihydrofolate reductase–thymidylate synthase

Abstract

The bifunctional enzyme dihydrofolate reductase–thymidylate synthase catalyses both the reductive methylation of 2′–deoxyuridylate and the subsequent reduction of dihydrofolate to yield 2′–deoxythymidylate and tetrahydrofolate at two spacially discrete sites situated on different protein domains. The X–ray structure of dihydrofolate reductase–thymidylate synthase from Leishmania major indicates that transfer of dihydrofolate between these sites does not occur by transient binding at both sites but rather by movement of dihydrofolate across the surface of the protein. The enzyme has an unusual surface charge distribution that could account for this channelling of dihydrofolate between active sites.

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References

  1. Ferone, R. & Roland, S. Dihydrofolate reductase:thymidylate synthase, a bifunctional polypeptide from Crithidia fasciculata. Proc. natn Acad. Sci. U.S.A. 77, 5802–5806 (1980).

    Article  CAS  Google Scholar 

  2. Ivanetich, K.M. & Santi, D.V. Bifunctional thymidylate synthase-dihydrofolate reductase in protozoa FASEB J. 4, 1591–1597 (1990).

    Article  CAS  Google Scholar 

  3. Cella, R., Carbonera, D., Orsi, R., Ferri, G. & ladarola, P. Proteolytic and partial sequencing studies of the bifunctional dihydrofolate reductase-thymidylate synthase from Daucus carota Plant molec. Biol. 16, 975–982 (1991).

    Article  CAS  Google Scholar 

  4. Lazar, G., Zhang, H. & Goodman, H.M. The origin of the bifunctional dihydrofolate reductase-thymidylate synthase isogenes of Arabidopsis thaliana Plant J. 3, 657–668 (1993).

    Article  CAS  Google Scholar 

  5. Meek, T.D., Garvey, E.P. & Santi, D.V. Purification and characterization of the bifunctional thymidylate synthase-dihydrofolate reductase from methotrexate-resistantle Leishmania tropica Biochemistry 24, 678–686 (1985).

    Article  CAS  Google Scholar 

  6. Kraut, J. & Matthews, D.A. in Biological Macromolecules and Assemblies, Vol. 3: Active Sites of Enzymes (eds Jurnak, F.A. & McPherson, A.) 1–71 (John Wiley & Sons, New York, 1987).

    Google Scholar 

  7. Bolin, J.T., Filman, D.J., Matthews, D.A., Hamlin, R.C. & Kraut, J. Crystal structures of Escherichia coli and Lactobacillus casei dihydrofolate reductase refined at 1.7 Å resolution: II General features and binding of methotrexate J. biol. Chem. 257, 13650–13662 (1982).

    CAS  PubMed  Google Scholar 

  8. Filman, D.J., Bolin, J.T., Matthews, D.A. & Kraut, J. Crystal structures of Escherichia coli and Lactobacillus casei dihydrofolate reductase refined at 1.7 Å resolution: II. Environment of bound NADPH and implications for catalysis J. biol. Chem. 257, 13663–13672 (1982).

    CAS  PubMed  Google Scholar 

  9. Davies, J.F., II et al. Crystal structures of recombinant human dihydrofolate reductase complexed with folate and 5-deazafolate Biochemistry 29, 9467–9479 (1990).

    Article  CAS  Google Scholar 

  10. Perry, K.M. et al. Plastic adaptation toward mutations in proteins: structural comparison of thymidylate synthases Proteins Struct. Funct. Genet. 8, 315–333 (1990).

    Article  CAS  Google Scholar 

  11. Hardy, L.W. et al. Atomic structure of thymidylate synthase: target for rational drug design Science 235, 448–455 (1987).

    Article  CAS  Google Scholar 

  12. Finer-Moore, J. et al. Refined structures of substrate-bound and phosphate-bound thymidylate synthase from Lactobacillus casei J. molec. Biol. 232, 1101–1116 (1993).

    Article  CAS  Google Scholar 

  13. Matthews, D.A., Appelt, K., Oatley, S.J. & Xuong, N.H. Crystal structure of Escherichia coli thymidylate synthase containing bound 5-fluoro-2′-deoxyuridylateand 10-propargyl-5,8-dideazafolate J. molec. Biol. 214, 923–936 (1990).

    Article  CAS  Google Scholar 

  14. Matthews, D.A. et al. Stereochemical mechanism of action for thymidylate synthase based on the X-ray structure of the covalent inhibitory ternary complex with 5-fluoro-2′-deoxyuridylate and 5, 10-methylenetetrahydrofolate J. molec. Biol. 214, 937–948 (1990).

    Article  CAS  Google Scholar 

  15. Montfort, W.R. et al. Structure, multiple site binding, and segmental accommodation in thymidylate synthase on binding dUMP and an anti-folate Biochemistry 29, 6964–6977 (1990).

    Article  CAS  Google Scholar 

  16. Perry, K.M., Carreras, C.W., Chang, L.C., Santi, D.V. & Stroud, R.M. Structures of thymidylate synthase with a C-terminal deletion: role of the C-terminus in alignment of 2′-deoxyuridine 5′-monophosphateand 5, 10-methylenetetrahydrofolate Biochemistry 32, 7116–7125 (1993).

    Article  CAS  Google Scholar 

  17. Ovadi, J. Physiological significance of metabolic channeling. J. theor. Biol. 152, 1–22 (1991).

    Article  CAS  Google Scholar 

  18. Anderson, K.S., Miles, E.W. & Johnson, K.A. Serine modulates substrate channeling in tryptophan synthase: a novel intersubunit triggering mechanism J. biol. Chem. 266, 8020–3033 (1991).

    CAS  PubMed  Google Scholar 

  19. Hyde, C.C., Ahmed, S.A., Padlan, E.A., Miles, E.W. & Davies, D.R. Three-dimensional structure of the tryptophan synthase α2β2 multienzyme complex from Salmonella typhimurium J. biol. Chem. 263, 17857–17871 (1988).

    CAS  PubMed  Google Scholar 

  20. Westerhoff, H.V. & Welch, G.R. Enzyme organization and the direction of metabolic flow: physicochemical considerations Curr. Topics cell. Reg. 33, 361–390 (1992).

    Article  CAS  Google Scholar 

  21. Koppenol, W.H. & Margoliash, E. The asymmetric distribution of charges on the surface of horse cytochrome c: functional implications J. biol. Chem. 257, 4426–4437 (1982).

    CAS  PubMed  Google Scholar 

  22. Ripoll, D.R., Faerman, C.H., Axelsen, P.H., Silman, I. & Sussman, J.L. An electrostatic mechanism for substrate guidance down the aromatic gorge of acetylcholinesterase Proc. natn Acad. Sci. U.S.A. 90, 5128–5132 (1993).

    Article  CAS  Google Scholar 

  23. Tan, R.C., Truong, T.N., McCammon, J.A. & Sussman, J.L. Acetylcholinesterase: electrostatic steering increases the rate of ligand binding Biochemistry 32, 401–403 (1993).

    Article  CAS  Google Scholar 

  24. Getzoff, E.D. et al. Electrostatic recognition between superoxide and copper, zinc superoxide dismutase Nature 306, 287–290 (1983).

    Article  CAS  Google Scholar 

  25. Getzoff, E.D. et al. Faster superoxide dismutase mutants designed by enhancing electrostatic guidance Nature 358, 347–351 (1992).

    Article  CAS  Google Scholar 

  26. McGuire, J.J. & Coward, J.K. in Folates and Pterins (eds Blakley, R.L. & Benkovic, S.J.) 135–190 (Wiley Interscience, New York, 1984).

    Google Scholar 

  27. Kisliuk, R.L. & Gaumont, Y. Polyglutamyl derivatives of folate as substrates and inhibitors of thymidylate synthetase J. biol. Chem. 249, 4100–4103 (1974).

    CAS  PubMed  Google Scholar 

  28. Kisliuk, R.L., Gaumont, Y., Baugh, C.M., Galivan, J.H., Maley, G.F., & Maley, F. in Chemistry and Biology of Pteridines (eds Kisliuk, R.L. & Brown, G.M.) 431–435 (Elsevier North Holland, New York, 1979).

    Google Scholar 

  29. Maley, G.F., Maley, F. & Baugh, C.M. Studies on identifying the folylpolyglutamate binding sites of Lactobacillus casei thymidylate synthetase Archs. Biochem. Biophys. 216, 551–558 (1982).

    Article  CAS  Google Scholar 

  30. Kamb, A., Finer-Moore, J., Calvert, A.H. & Stroud, R. Structural basis for recognition of polyglutamyl folates by thymidylate synthase Biochemistry 41, 9883–9890 (1993).

    Google Scholar 

  31. Roos, D.S. Primary structure of the dihydrofolate reductase-thymidylate synthase gene from Toxoplasma gondii J. biol. Chem. 268, 6269–6280 (1993).

    CAS  PubMed  Google Scholar 

  32. Beverley, S.M., Ellenberg, T.E., Cordingley, J.S. Primary structure of the gene encoding the bifunctional dihydrofolate reductase-thymidylate synthase of Leishmania major Proc. natn. Acad. Sci U.S.A. 83, 2584–2588 (1986).

    Article  CAS  Google Scholar 

  33. Kunkel, T.A. Rapid and efficient site-specific mutagenesis without phenotypic selection Proc. natn. Acad. Sci. U.S.A. 82, 488–492 (1985).

    Article  CAS  Google Scholar 

  34. Ghrayeb, J. et al. Secretion cloning vectors in Escherichia coli EMBO J. 3, 2437–2442 (1984).

    Article  CAS  Google Scholar 

  35. Brunger, A.T., Kuriyan, J. & Karplus, M. Crystallographic R factor refinement by molecular dynamics Science 235, 458–460 (1987).

    Article  CAS  Google Scholar 

  36. Read, R. Improved Fourier coefficients for maps using phases from partial structures with errors Acta crystallogr. A42, 140–149 (1986).

    Article  CAS  Google Scholar 

  37. Bernstein, F.C. et al. The Protein Data Bank: a computer-based archival file for macromolecular structures J. molec. Biol. 112, 535–542 (1977).

    Article  CAS  Google Scholar 

  38. Tronrud, D.E. Conjugate-direction minimization—an improved method for the refinement of macromolecules Acta crystallogr. A48, 912–916 (1992).

    Article  CAS  Google Scholar 

  39. Tronrud, D.E., Ten Eyck, L.F. & Matthews, B.W. An efficient general-purpose least-squares refinement program for macromolecular structures Acta crystallogr. A43, 489–501 (1987).

    Article  CAS  Google Scholar 

  40. Jones, T.A. A graphics model building and refinement system for macromolecules J. appl. Crystallogr. 11, 268–272 (1978).

    Article  CAS  Google Scholar 

  41. Weiner, S.J. et al. A new force field for molecular mechanical simulation of nucleic acids and proteins J. Am. chem. Soc. 106, 765–784 (1984).

    Article  CAS  Google Scholar 

  42. Gilson, M.K., Sharp, K.A. & Honig, B.H. Calculating the electrostatic potential of molecules in solution: method and error assessment J. comput. Chem. 9, 327–335 (1987).

    Article  Google Scholar 

  43. Jayaram, B., Sharp, K. & Honig, B. The electrostatic potential of B-DNA Biopolymers 28, 975–993 (1989).

    Article  CAS  Google Scholar 

  44. Kraulis, P.J. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures J. appl. Crystallogr. 24, 946–950 (1991).

    Article  Google Scholar 

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Knighton, D., Kan, CC., Howland, E. et al. Structure of and kinetic channelling in bifunctional dihydrofolate reductase–thymidylate synthase. Nat Struct Mol Biol 1, 186–194 (1994). https://doi.org/10.1038/nsb0394-186

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